Investigating stability landscape of Aurora kinase B probed by guanidinium chloride-induced unfolding

Aurora kinase B (AURKB), a serine/threonine kinase, is a key mitotic regulator essential for chromosome segregation, cytokinesis, and orderly cell division. Structural instability of AURKB can disrupt mitotic progression and, in turn, promote oncogenic transformation. In this study, we examined the structural stability landscape of human AURKB using guanidinium chloride (GdmCl)-induced denaturation. Structural perturbations in AURKB were assessed using far-UV circular dichroism (CD) and intrinsic tryptophan fluorescence spectroscopy. Spectroscopic analyses revealed a concentration-dependent destabilization of AURKB, with partial unfolding at 1.0 M GdmCl and significant loss of structure at 2.0 M GdmCl. Although AURKB retained residual activity at 2.0-2.5 M, complete loss was observed at higher concentrations. The stability parameters, including Gibbs free energy in the absence of denaturant (ΔG⁰_D), the midpoint of denaturation (C_m), and the slope (m) of the ΔG_D versus [GdmCl] plot, were calculated from the unfolding curves. The overlapping transition profiles indicated a two-state unfolding mechanism (N ⇌ D). Enzymatic assays further showed that even low GdmCl concentrations attenuated AURKB catalytic activity. MD simulations were performed to investigate the early steps of protein unfolding. Both complementary biophysical and computational analyses delineate the unfolding landscape of AURKB, offering mechanistic insight into the structural determinants governing kinase stability. This work enhances the understanding of AURKB behaviour under non-native conditions and may inform the rational design of stabilization strategies for therapeutic applications.